Proteins are linear chains of twenty covalently linked naturally occurring amino acids. The amino acid sequence or primary structure determines the manner in which the chain can fold to form the secondary and tertiary structures necessary for biological function. When only smaller quantities of a rare protein are isolated sequence analysis is an essential prerequisite to cloning.
Currently, protein sequence analysis is primarily accomplished with the use of an automated sequencer using chemistry developed by Edman over 40 years ago (Edman, Acta Chem. Scand. 4:283-293 (1950)). Since that time improvement in the instrumentation has resulted in the ability to sequence smaller and smaller sample quantities (mmole to pmol), although the original chemistry has remained essentially unchanged. Current automated instrumentation permits 10-20 cycles of sequence determination on 10-50 pmol of sample (Simpson, et al., Anal. Biochem. 177:221-236 (1989)).
Advances in protein isolation methodology have recently made it possible to isolate sub-picomole quantities of proteins of biological interest which are present in tissues. Improved methods of protein sequencing requiring less sample quantity would make it possible to obtain the necessary sequence information in order to clone and express these proteins, and hence to study the structure function aspects thereof. These proteins often have key roles in the development and treatment of human disease.
The major limitation of Edman chemistry is the practical detection limit of the peptidylthiohydantoin (PTH) amino acids. The current method involves separation of the PTH amino acids by high-performance liquid chromatograph (HPLC) using UV detection. The practical detection limit of this method is approximately 1 pmol. A number of methods have been proposed to increase the sensitivity of Edman degradation by the use of radiolabeled, chromophoric, or fluorescent isothiocyanate reagents. 4-(N,N'-Dimethylamino)azobenzene-4'-isothiocyanate (DABITC), a highly chromophoric reagent, was introduced by Chang (Chang, et al., Biochem. J. 153:607-611 (1976)). Fluorescent reagents, such as fluorescein isothiocyanate (Maeda, et al. Biochem. Biophys. Res. Commun. 31:188-192 (1968); Muramoto, K., et al. Anal. Biochem. 141:446-450 (1984)), and dansyl-containing isothiocyanates (Hirano, et al. Biol. Chem. Hoppe-Seyler 164:257-263 (1986); Hirano, et al. "Methods in Protein Sequence Analysis" (Ed. B. Wittman-Liebold) Springer-Verlag, Berlin, pp. 42-51 (1986); Jin, S. W., et al. FEBS Lett. 198:105-154 (1986); Jin, S. W., et al., In: Methods in Protein Sequence Analysis (Ed. B. Wittman-Liebold) Springer-Verlag, Berlin, pp. 34-41 (1989); Sainikow, J., et al. In: Methods in Protein Sequence Analysis (Ed. K. A. Walsh) Humana Press, Clifton, N.J., pp. 247-260 (1987)) have also been evaluated as sensitivity enhancing reagents. Although synthetically prepared amino acid analogues prepared using these reagents have shown subpicomole sensitivity by HPLC analysis, in automated sequencing the sensitivity of the standard Edman methodology has not been surpassed. It is postulated that the large chromophore of such reagents may interfere with the derivatization and cleavage reactions of the Edman degradation. The use of radiolabeled reagents has failed consequent from autoradiodegradation which results in decreasing product yields and increasing amounts of labeled by-products.
An alternative method has more recently been proposed which involves treatment of the anilinothiazolinone (ATZ) derivative normally formed in Edman chemistry with a fluorescent amine (Tsugita, A., et al., J.Biochem. 106:60-65 (1989)). The advantage of this approach is that the derivatization and cleavage reactions of the Edman chemistry remain unchanged. Theoretically this chemistry should permit sequencing on femtomole levels of sample. However, investigation of this has revealed a number of problems which tend to defeat the goal of more sensitive sequencing. Foremost is the instability of the ATZ-amino acids which are required for reaction with the fluorescent amine. The ATZ-amino acids, in particular the hydrophilic amino acids such as histidine, glutamate, and aspartate, were found to rearrange to the PTH derivative so rapidly that reaction with the fluorescent amine was not possible.
A possible solution to this problem is to convert the PTH amino acid back to the ATZ amino acid so that reaction with the fluorescent amine will be possible. The aminolysis of PTH amino acids is discussed in detail by Pavlik, et al., Anal. Biochem. 201:9-16 (1992). Replacement of the fluorescent amine with a reagent such as N,N-dimethylethylenediamine (DMED) has been found to permit detection in the femtomole level using electrospray mass spectrometry. The introduction of the tertiary amine to the amino acid derivative was found to enhance detection of the amino acid by 25 times as compared to the PTH-amino acid, thereby making mass spectrometry a viable method for enhancing the levels of detection during protein sequencing.